Muscarinic receptor agonists activate phosphoinositide hydrolysis and adenylate cyclase in Chinese hamster ovary cells transfected with cDNAs encoding the human muscarinic ml and m3 receptors. Whereas carbachol activates similarly both receptor subtypes, 4-[3-chlorophenyl-carbamoyloxy]-2-butynyltrimethyl ammonium chloride (McN-A-343) preferentially activates the m1 subtype over m3, in regard to both phosphoinositide hydrolysis and adenylate cyclase activity. On the other hand, oxotremorine activates phosphoinositide hydrolysis to a similar extent in both cell lines, but it activates preferentially adenylate cyclase in m1 versus m3 receptor expressing cells. Relative to carbachol, both McN-A-343 and oxotremorine activate preferentially phosphoinositide hydrolysis over adenylate cyclase in both cell lines. Prolonged incubation of cells with either carbachol, McN-A-343, or oxotremorine down-regulated the m1 receptors. This was accompanied by a parallel decrease in adenylate cyclase activity, whereas phosphoinositide hydrolysis remained relatively high. Inactivation of the receptors by alkylation with acetylethylcholine mustard, or by blocking with atropine, reduced carbachol-stimulated adenylate cyclase activity more effectively than carbachol-induced phosphoinositide hydrolysis in both m1 and m3 receptor expressing cells. These findings imply that the receptor reserve in these cell lines is greater for phosphoinositide hydrolysis response than for adenylate cyclase response. Yet, the receptor reserve for each of these responses is similar in both m1 and m3 receptor expressing cells. Since the binding affinities of McN-A-343 and of oxotremorine to m1 and m3 receptors are very similar, and since both cell lines contain similar amounts of spare receptors, we propose that the preferential activation of muscarinic m1 over m3 receptor by partial agonists is related to differences in the abilities of the two receptor subtypes to undergo conformational changes following agonist binding. This hypothesis is supported by results showing that the muscarinic m1 but not m3 receptor exhibits two affinity states in a competition binding assay.